Novel genetic products obtained from ashbya gossypii, which are associated with transcription mechanisms, rna processing and/or translation

ABSTRACT

The invention relates to novel polynucleotides from  Ashbya gossypii , to oligonucleotides hybridizing therewith; to expression cassettes and vectors which comprise these polynucleotides; to microorganisms transformed therewith; to polypeptides encoded by these polynucleotides; and to the use of the novel polypeptides and polynucleotides as targets for modulating transcription and/or translation control and/or control of RNA processing and, in particular, improving vitamin B2 production in microorganisms of the genus  Ashbya.

The present invention relates to novel polynucleotides from Ashbyagossypii; to oligonucleotides hybridizing therewith; to expressioncassettes and vectors which comprise these polynucleotides; tomicroorganisms transformed therewith; to polypeptides encoded by thesepolynucleotides; and to the use of the novel polypeptides andpolynucleotides as targets for modulating the operations oftranscription, RNA processing and/or translation and, in particular,improving vitamin B2 production in microorganisms of the genus Ashbya.

Vitamin B2 (riboflavin, lactoflavin) is an alkali- and light-sensitivevitamin which shows a yellowish green fluorescence in solution. VitaminB2 deficiency may lead to ectodermal damage, in particular cataract,keratitis, corneal vascularization, or to autonomic and urogenitaldisorders. Vitamin B2 is a precursor for the molecules FAD and FMNwhich, besides NAD⁺ and NADP⁺, are important in biology for hydrogentransfer. They are formed from vitamin B2 by phosphorylation (FMN) andsubsequent adenylation (FAD).

Vitamin B2 is synthesized in plants, yeasts and many microorganisms fromGTP and ribulose 5-phosphate. The reaction pathway starts with openingof the imidazole ring of GTP and elimination of a phosphate residue.Deamination, reduction and elimination of the remaining phosphate resultin 5-amino-6-ribitylamino-2,4-pyrimidinone. Reaction of this compoundwith 3,4-dihydroxy-2-butanone 4-phosphate leads to the bicyclic molecule6,7-dimethyl-8-ribityllumazine. This compound is converted into thetricyclic compound riboflavin by dismutation, in which a 4-carbon unitis transferred.

Vitamin B2 occurs in many vegetables and in meat, and to a lesser extentin cereal products. The daily vitamin B2 requirement of an adult isabout 1.4 to 2 mg. The main breakdown product of the coenzymes FMN andFAD in humans is in turn riboflavin, which is excreted as such.

Vitamin B2 is thus an important dietary substance for humans andanimals. Efforts are therefore being made to make vitamin B2 availableon the industrial scale. It has therefore been proposed to synthesizevitamin B2 by a microbiological route. Microorganisms which can be usedfor this purpose are, for example, Bacillus subtilis, the ascomycetesEremothecium ashbyii, Ashbya gossypii, and the yeasts Candida flareriand Saccharomyces cerevisiae. The nutrient media used for this purposecomprise molasses or vegetable oils as carbon source, inorganic salts,amino acids, animal or vegetable peptones and proteins, and vitaminadditions. In sterile aerobic submerged processes, yields of more than10 g of vitamin B2 are obtained per liter of culture broth within a fewdays. The requirements are good aeration of the culture, carefulagitation and setting of temperatures below about 30° C. Removal of thebiomass, evaporation and drying of the concentrate result in a productenriched in vitamin B2.

Microbiological production of vitamin B2 is described, for example, inWO-A-92101060, EP-A-0 405 370 and EP-A-0 531 708.

A survey of the importance, occurrence, production, biosynthesis and useof vitamin B2 is to be found, for example, in Ullmann's Encyclopaedia ofIndustrial Chemistry, volume A27, pages 521 et seq.

Transcription

Gene expression in fungi is mainly controlled at the transcriptionlevel. The transcription apparatus consists of a number of proteinswhich can be divided into two groups: RNA polymerase (the operativeDNA-transcribing enzyme) and transcription factors (which control genetranscription by guiding the RNA polymerase to specific promoter DNAsequences which recognize these factors). Fungi such as Ashbya gossypiicontain a number of different transcription factors which are specificfor different promoters, growth phases, environmental conditions,substrates, oxygen levels and the like, allowing the organism to adaptto various environmental and metabolic conditions.

Promoters are specific DNA sequences which serve as docking sites forthe RNA polymerase complex and transcription factors. Many promoterelements have conserved sequence elements which can be identified byhomology searches; an alternative possibility is to identify promoterregions for a particular gene using standard techniques such as primerextension. Many promoter regions of eukaryotes are known (Guarente, L(1987), Ann. Rev. Biochem., 21; 425-452).

Promoter transcription control is influenced by several repression oractivation mechanisms. Specific regulatory proteins (transcriptionfactors), which bind to promoters, have the ability to block the bindingof the RNA holoenzyme (repressors) or assist the latter (activators) andthus control transcription. In addition, certain enzymes modify thehistones bound to the DNA and thus make it possible for either access ofthe transcription factors to the promoter to be prevented or madepossible for the first time (Loo, S.; Rine, J. (1995); Annu. Rev. Cell.Dev. Biol., 11, 519-548). The binding of the transcription factors islikewise controlled by their interactions with other molecules such asproteins or other metabolic compounds (Evans, R. (1989), Science, 240,889-895). The ability to control the transcription of genes thusresponds to a plurality of environmental or metabolic signs makes itpossible for the cells to control exactly when a gene can be expressedand how much of a gene product can be present in the cell at a point intime. This in turn prevents unnecessary expenditure of energy orunnecessary use of possibly rare intermediate compounds or cofactors.

RNA Processing

RNA is synthesized as heterogeneous fragment, with the coding sequence(exons) in eukaryotes frequently being interrupted by noncodingsequences (introns). During RNA processing after transcription, theintrons are cut out (splicing) so that the coding sequence (of mRNA) canbe read off on the ribosomes (Sharp, P. (1987), Science; 235, 766-771).Since the export of RNA from the cell is also controlled with thesplicing, it is possible in this way to control the amount of mRNAavailable on the ribosomes.

Translation

Translation is the process by which a polypeptide is synthesized fromamino acids in accordance with the information contained in an RNAmolecule. The main components of this process are ribosomes and specificinitiation or elongation factors such as eEF1 and eEF2 (Moldave (1985);Ann. Rev. Biochem., 54, 1109-1149). Ribosomes are composed of RNA (rRNA)and specific proteins. They consist of a large and a small subunit, eachof which can be characterized by its sedimentation behavior in ananalytical ultracentrifuge. Synthesis of the ribosomes is controlled bycoordinated production of the RNA and protein components depending onthe physiological state of the cell.

Each codon of the mRNA molecule encodes a particular amino acid. Theconversion of mRNA into amino acid is carried out by transfer RNA (tRNA)molecules. These molecules consist of an RNA single strand (between 60and 100 bases) which is in the form of an L-shaped three-dimensionalstructure with projecting regions or “arms”. One of these arms formsbase pairs with a particular codon sequence on the mRNA molecule. Asecond arm interacts specifically with a particular amino acid (which isencoded by the codon). Other tRNA arms comprise the variable arm, theTΨC arm (which has thymidylate and pseudouridylate modifications) andthe D arm (which has a dihydrouridine modification). The function ofthese latter structures is still unknown, but their conservation betweentRNA molecules suggests a role in protein synthesis.

In order that the nucleic acid-based tRNA molecule pairs with thecorrect amino acid it is necessary for a family of enzymes, referred toas aminoacyl-tRNA synthetases, to act. There are many different enzymesof this type, and each is specific for a particular tRNA and aparticular amino acid. These enzymes bind the 3′-hydroxyl of theterminal tRNA adenosine ribose unit to the amino acid in a two-stepreaction. Firstly, the enzyme is activated by reaction with ATP and theamino acid, resulting in an aminoacyl-tRNA synthetase/aminoacyladenylate complex. Secondly, the aminoacyl group is transferred from theenzyme to the target tRNA, on which it remains in a high-energy state.Binding of the tRNA molecule to its recognition codon on the mRNAmolecule then brings the high-energy amino acid bound to the tRNA intocontact with the ribosome. Inside the ribosome, the amino acid-loadedtRNA (aminoacyl-tRNA) occupies a binding site (the A site) next to asecond site (the P site) which carries a tRNA molecule whose amino acidis bound to the growing polypeptide chain (peptidyl-tRNA). The activatedamino acid on the aminoacyl-tRNA is sufficiently reactive for a peptidebond to form spontaneously between this amino acid and the next aminoacid on the growing polypeptide chain. GTP hydrolysis supplies theenergy to transfer the tRNA, which is now loaded with the polypeptidechain, from the A site to the P site of the ribosome, and the process isrepeated until a stop codon is reached.

There is a number of different steps at which translation can becontrolled. These include binding of the ribosome to mRNA, the presenceof mRNA secondary structure, the codon usage or the frequency ofparticular tRNAs.

The utilization of genes associated with the mechanisms oftranscription, RNA processing and/or translation for generatingmicroorganisms, preferably of the genus Ashbya, in particular of Ashbyagossypii strains, with improved adaptability to external conditions suchas environmental and metabolic conditions has not yet been described.

It is an object of the present invention to provide novel targets forinfluencing the transcription and/or translation mechanisms and/or themechanisms of RNA processing in microorganisms of the genus Ashbya, inparticular in Ashbya gossypii. The object in particular is specificmodulation of the transcription, RNA processing and/or translation insuch microorganisms. A further object is to improve the vitamin B2production by such microorganisms.

We have found that this object is achieved by providing encoding nucleicacid sequences which are upregulated or downregulated in Ashbya gossypiiduring vitamin B2 production (based on results found with the aid of theMPSS analytical method described in detail in the experimental part), inparticular a) a, preferably upregulated, nucleic acid sequence whichcodes for a protein having the function of a 26 S proteasome subunit orof a TAT binding homolog 7.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 28”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 28v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 1. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 4 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 28” and “Oligo 28v” have significant homologieswith the MIPS tag “Yta7” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4. The amino acidsequences derived therefrom have significant sequence homology with a 26S proteasome subunit or a TAT-binding homolog 7 (TBP-7) from S.cerevisiae.

b) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a translation initiation factor subunit.

In a preferred embodiment there has been isolation according to theinvention of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 45”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 45v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 6. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 10 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 45” and “Oligo 45v” have significant homologieswith the MIPS tag “p39” or “Tif34” from S. cerevisiae. The inserts havea nucleic acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 10. Anamino acid sequence derived therefrom has significant sequence homologywith a subunit (P39) of the translation initiation factor EIF3 (IF32)from S. cerevisiae.

c) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of a ribosomal protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 85”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 85v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 12. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 14 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 85” and “Oligo 85v” have significant homologieswith the MIPS tag “Rpl35a” from S. cerevisiae. The inserts have anucleic acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 14. Theamino acid sequence derived from the coding strand or amino acidpart-sequence has significant sequence homology with a ribosomal proteinfrom S. cerevisiae.

d) a, preferably upregulated, nucleic acid sequence codes for a proteinhaving the function of a nucleolar protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 133”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 133v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 17. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 19 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 133” and “Oligo 133v” have significant homologieswith the MIPS tag “Nop13” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 19. The amino acidsequence or amino acid part-sequence derived from the correspondingcomplementary strand of SEQ ID NO: 17 or from the sequence shown in SEQID NO: 19 has significant sequence homology with a nucleolar proteinfrom S. cerevisiae.

e) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a translation initiation protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 172”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 172v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 21. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 24 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 172” and “Oligo 172v” have significant homologieswith the MIPS tag “Sua5” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 24. The amino acidsequence or amino acid part-sequence derived from the coding strand hassignificant sequence homology with a translation initiation protein fromS. cerevisiae.

f) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of a precursor of ribosomal protein S 31.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 63”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 63v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 26. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 29 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 63” and “Oligo 63v” have significant homologieswith the MIPS tag “Rps25a” from S. cerevisiae. The inserts have anucleic acid sequence as shown in SEQ ID NO: 26 or SEQ ID NO: 29. Theamino acid sequence or amino acid part-sequence derived from thecorresponding complementary strand of SEQ ID NO: 26 or from the codingstrand shown in SEQ ID NO: 29 has significant sequence homology with aprecursor of the ribosomal protein S 31 from S. cerevisiae.

g) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of a cell nuclear pore protein.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 132”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 132v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 31. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 36 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 132” and “Oligo 132v” have significant homologieswith the MIPS tag “Nic96” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 31 or SEQ ID NO: 36. An amino acidsequence derived therefrom (corresponding to nucleotides 108 to 764 inSEQ ID NO: 31) has significant sequence homology with a cell nuclearpore protein from S. cerevisiae.

h) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a constituent of the ADH-histoneacetyltransferase complex.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 174”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 174v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 38. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 40 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 174” and “Oligo 174v” have significant homologieswith the MIPS tag “Ahc1” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 38 or SEQ ID NO: 40. The amino acidsequence or amino acid part-sequence derived from the correspondingcomplementary strand to SEQ ID NO: 38 or from the coding strand shown inSEQ ID NO: 40 has significant sequence homology with a constituent ofthe ADH-histone acetyltransferase complex from S. cerevisiae.

i) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of an RNA helicase involved in RNAprocessing.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 51”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 51v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 42. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 46 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 51” and “Oligo 51v” have significant homologieswith the MIPS tag “Rok1” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 42 or SEQ ID NO: 46. The amino acidsequence derived from the corresponding complementary strand to SEQ IDNO: 42 or from the coding strand of SEQ ID NO: 46 have significantsequence homology with a S. cerevisiae RNA helicase involved in RNAprocessing.

k) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of the non-essential constituent of RNApoll.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 30”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 30v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 48. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 51 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 30” and “Oligo 30v” have significant homologieswith the MIPS tag “Rpa34” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 48 or SEQ ID NO: 51. The amino acidsequences derived in each case from the coding strand have significantsequence homology with the non-essential constituent of RNA poll from S.cerevisiae.

l) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of an RNA helicase.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 124”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 124v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 53. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 56 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 124” and “Oligo 124v” have significant homologieswith a MIPS tag “Sub2” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 53 or SEQ ID NO: 56. The amino acidsequence or amino acid part-sequence derived from the coding strand hassignificant sequence homology with an RNA helicase from S. cerevisiae.

m) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of an mRNA decapping enzyme.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 139”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 139v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 58. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 60 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 139” and “Oligo 139v” have significant homologieswith the MIPS tag “DCP1” from S. cerevisiae. The inserts having nucleicacid sequence as shown in SEQ ID NO: 58 or SEQ ID NO: 60. The amino acidsequence or amino acid part-sequence derived from the coding strand hassignificant sequence homology with an mRNA decapping enzyme from S.cerevisiae.

n) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of a subunit of the translation initiationfactor eIF3.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 144”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 144v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 63. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 65 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 144” and “Oligo 144v” have significant homologieswith the MIPS tag “PRT1” from S. cerevisiae. The inserts have a nucleicacid sequence as shown in SEQ ID NO: 63 or SEQ ID NO: 65. The amino acidsequence or amino acid part-sequence derives from the coding strand assignificant sequence homology with a subunit of the translationinitiation factor eIF3 from S. cerevisiae.

o) a, preferably upregulated, nucleic acid sequence which codes for aprotein having the function of a U3 small nucleolarribonucleoprotein-substituted protein which is involved in preribosomalRNA processing.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 168”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 168v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 67. A furtheraspect of the invention relates to a polynucleotide comprising a nucleicacid sequence as shown in SEQ ID NO: 70 or a fragment thereof. Thepolynucleotides can be isolated preferably from a microorganism of thegenus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

The inserts of “Oligo 168” and “Oligo 168v” have significant homologieswith the MIPS tag “Rrp9” from S. cerevisiae. The inserts have thenucleic acid sequence as shown in SEQ ID NO: 67 or SEQ ID NO: 70. Theamino acid sequence or amino acid part-sequence derived from the codingstrand has significant sequence homology with a S. cerevisiae U3 smallnucleolar ribonucleoprotein-associated protein which is involved inpreribosomal RNA processing.

p) a, preferably downregulated, nucleic acid sequence which codes for aprotein having the function of the ribosomal protein L7a.e.B of thelarge 60 S subunit.

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 160”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 72, which canbe isolated preferably from a microorganism of the genus Ashbya, inparticular A. gossypii. The invention additionally relates to thepolynucleotide complementary thereto; and to the sequences derived fromthese polynucleotides through the degeneracy of the genetic code.

The insert of “Oligo 160” has significant homologies with the MIPS tag“Rpl8b” from S. cerevisiae. The insert has a nucleic acid sequence asshown in SEQ ID NO: 72. The amino acid sequence derived from thecorresponding complementary strand has significant sequence homologywith a ribosomal protein (L7a.e.B; large 60S subunit) from S.cerevisiae.

q) We have found that this object is achieved by providing an encodingnucleic acid sequence which is unregulated in Ashbya gossypii duringvitamin B2 production (based on results found with the aid of the MPSSanalytical method described in detail in the experimental part).

In a preferred embodiment of this aspect of the invention there has beenisolation of a DNA clone which codes for a characteristic part-sequenceof the nucleic acid sequence of the invention and which bears theinternal name “Oligo 18”.

In a further preferred embodiment there has been isolation according tothe invention of a DNA clone which codes for the complete sequence ofthe nucleic acid of the invention and which bears the internal name“Oligo 18v”.

A first aspect of the present invention relates to a polynucleotidecomprising a nucleic acid sequence as shown in SEQ ID NO: 75 or thepolynucleotide complementary thereto as shown in SEQ ID NO: 74. Afurther aspect of the invention relates to a polynucleotide comprising anucleic acid sequence as shown in SEQ ID NO: 77 or a fragment thereof.The polynucleotides can be isolated preferably from a microorganism ofthe genus Ashbya, in particular A. gossypii. The invention additionallyrelates to the polynucleotides complementary thereto; and to thesequences derived from these polynucleotides through the degeneracy ofthe genetic code.

A further aspect of the invention relates to oligonucleotides whichhybridize with one of the above polynucleotides, in particular understringent conditions.

The invention additionally relates to polynucleotides which hybridizewith one of the oligonucleotides of the invention and code for a geneproduct from microorganisms of the genus Ashbya or a functionalequivalent of this gene product.

The invention further relates to polypeptides or proteins which areencoded by the polynucleotides described above; and to peptide fragmentsthereof which have an amino acid sequence which comprises at least 10consecutive amino acid residues as shown in SEQ ID NO: 2, 3, 5, 7, 8, 9,11, 13, 15, 16, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 34, 35, 37, 39,41, 43, 44, 45, 47, 49, 50, 52, 54, 55, 57, 59, 61, 62, 64, 66, 68, 69,71, 73, 76, or SEQ ID NO: 78; and to functional equivalents of thepolypeptides or proteins of the invention.

In this connection, functional equivalents differ from the productsspecifically disclosed in the invention by their amino acid sequencethrough addition, insertion, substitution, deletion or inversion at aminimum of one, such as, for example, 1 to 30 or 1 to 20 or 1 to 10,sequence positions without the originally observed protein function,which can be deduced by sequence comparison with other proteins, beinglost. It is thus possible for equivalents to have essentially identical,higher or lower activities compared with the native protein.

Further aspects of the invention relate to expression cassettes for therecombinant production of proteins of the invention, comprising one ofthe nucleic acid sequences defined above, operatively linked to at leastone regulatory nucleic acid sequence; and to recombinant vectorscomprising at least one such expression cassette of the invention.

Also provided according to the invention are prokaryotic or eukaryotichosts which are transformed with at least one vector of the above type.A preferred embodiment provides prokaryotic or eukaryotic hosts in whichthe functional expression of at least one gene which codes for apolypeptide of the invention as defined above is modulated (e.g.inhibited or overexpressed); or in which the biological activity of apolypeptide as defined above is reduced or increased. Preferred hostsare selected from ascomycetes, in particular those of the genus Ashbyaand preferably strains of A. gossypii.

Modulation of gene expression in the above sense includes bothinhibition thereof, for example through blockade of a stage inexpression (in particular transcription or translation) or a specificoverexpression of a gene (for example through modification of regulatorysequences or increasing the copy number of the coding sequence).

A further aspect of the invention relates to the use of an expressioncassette of the invention, of a vector of the invention or of a host ofthe invention for the microbiological production of vitamin B2 and/orprecursors and/or derivatives thereof.

A further aspect of the invention relates to the use of an expressioncassette of the invention, of a vector of the invention or of a host ofthe invention for the recombinant production of a polypeptide of theinvention as defined above.

Also provided according to the invention is a method for detecting orfor validating an effector target for modulating the microbiologicalproduction of vitamin B2 and/or precursors and/or derivatives thereof.This entails treating a microorganism capable of the microbiologicalproduction of vitamin B² and/or precursors and/or derivatives thereofwith an effector which interacts with (such as, for example,non-covalently binds to) a target selected from a polypeptide of theinvention as defined above or a nucleic acid sequence coding therefor,validating the influence of the effector on the amount of themicrobiologically produced vitamin B2 and/or of the precursor and/or ofa derivative thereof; and isolating the target where appropriate. Thevalidation in this case takes place preferably by direct comparison withthe microbiological vitamin B2 production in the absence of the effectorunder otherwise identical conditions.

A further aspect of the invention relates to a method for modulating (inrelation to the amount and/or rate of) the microbiological production ofvitamin B2 and/or precursors and/or derivatives thereof, where amicroorganism capable of the microbiological production of vitamin B2and/or precursors and/or derivatives thereof is treated with an effectorwhich interacts with a target selected from a polypeptide of theinvention as defined above or a nucleic acid sequence coding therefor.

Preferred examples of the abovementioned effectors which should bementioned are:

-   a) antibodies or antigen-binding fragments thereof;-   b) polypeptide ligands which are different from a) and which    interact with a polypeptide of the invention;-   c) low molecular weight effectors which modulate the biological    activity of a polypeptide of the invention;-   d) antisense nucleic acid sequences which interact with a nucleic    acid sequence of the invention.

The invention likewise relates to the abovementioned effectors havingspecificity for at least one of the targets, according to the invention,defined above.

A further aspect of the invention relates to a method for themicrobiological production of vitamin B2 and/or precursors and/orderivatives thereof, where a host as defined above is cultivated underconditions favoring the production of vitamin B2 and/or precursorsand/or derivatives thereof, and the desired product(s) is(are) isolatedfrom the culture mixture. It is preferred in this connection that thehost is treated with an effector as defined above before and/or duringthe cultivation. A preferred host is in this case selected frommicroorganisms of the genus Ashbya; in particular transformed asdescribed above.

A final aspect of the invention relates to the use of a polynucleotideor polypeptide of the invention as target for modulating the productionof vitamin B2 and/or precursors and/or derivatives thereof in amicroorganism of the genus Ashbya.

DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment between an amino acid sequence of theinvention based on SEQ ID NO: 5 (middle sequence) and a part-sequence ofthe MIPS tag “Yta7” from S. cerevisiae (lower sequence). The consensussequence is depicted above these two. Positions lacking homology aresymbolized by black rectangles.

FIG. 2 shows an alignment between an amino acid sequence of theinvention based on SEQ ID NO: 11 (middle sequence) and a part-sequenceof the MIPS tag “Tif34” from S. cerevisiae (lower sequence). Theconsensus sequence is depicted above these two. Positions lackinghomology are symbolized by black rectangles.

FIG. 3 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 469 to 825 in SEQ IDNO: 12 (upper sequence) and a part-sequence of the MIPS tag “Rpl25a”from S. cerevisiae (lower sequence) Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 4 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand at position 114 to1 in SEQ ID NO: 17 (upper sequence) and a part-sequence of the MIPS tag“Nopl3” from S. cerevisiae (lower sequence). Identical sequencepositions are indicated between the two sequences. Similar sequencepositions are labeled with “+”.

FIG. 5A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 2 to 349 in SEQ IDNO: 21) (upper sequence) and a part-sequence of the MIPS tag “Sua5” fromS. cerevisiae (lower sequence). FIG. 5B shows an alignment between anamino acid part-sequence of the invention (corresponding to the strandat position 336 to 947 in SEQ ID NO: 21) (upper sequence) and apart-sequence of the MIPS tag “Sua5” from S. cerevisiae (lowersequence).

FIG. 6A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 609 to562 in SEQ ID NO: 26) (upper sequence) and a part-sequence of the MIPStag “Rps25a” from S. cerevisiae (lower sequence). FIG. 6B shows analignment between an amino acid part-sequence of the invention(corresponding to the complementary strand to position 556 to 401 in SEQID NO: 26) (upper sequence) and a part-sequence of the MIPS tag “Sua5”from S. cerevisiae (lower sequence). Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 7 shows an alignment between an amino acid sequence of theinvention based on SEQ ID NO: 36 (middle sequence) and a part-sequenceof the MIPS tag “Nic96” from S. cerevisiae (lower sequence). Theconsensus sequence is depicted above these two. Positions lackinghomology are symbolized by black rectangles.

FIG. 8 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand at position 174 to1 in SEQ ID NO: 38) (upper sequence) and a part-sequence of the MIPS tag“Ahcl” from S. cerevisiae (lower sequence). Identical sequence positionsare indicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 9A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand to position 1086 to1012 in SEQ ID NO: 42) (upper sequence) and a part-sequence of the MIPStag “Rok1” from S. cerevisiae (lower sequence). FIG. 9B shows analignment between an amino acid part-sequence of the invention(corresponding to the complementary strand to position 1022 to 915 inSEQ ID NO: 42) (upper sequence) and a part-sequence of the MIPS tag“Rok1” from S. cerevisiae (lower sequence). FIG. 9C shows an alignmentbetween an amino acid part-sequence of the invention (corresponding tothe complementary strand to position 925 to 689 in SEQ ID NO: 42) (uppersequence) and a part-sequence of the MIPS tag “Rok1” from S. cerevisiae(lower sequence). Identical sequence positions are in each caseindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 10A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 1 to 102 in SEQ IDNO: 48) (upper sequence) and a part-sequence of the MIPS tag “Rpa43”from S. cerevisiae (lower sequence). FIG. 10B shows an alignment betweenan amino acid part-sequence of the invention (corresponding to thestrand at position 122 to 400 in SEQ ID NO: 48) (upper sequence) and apart-sequence of the MIPS tag “Rpa43” from S. cerevisiae (lowersequence). Identical sequence positions are indicated between the twosequences. Similar sequence positions are labeled with “+”. FIG. 10Cshows the coding part-sequence as shown in SEQ ID NO: 48 and thepart-sequence complementary thereto.

FIG. 11A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 2 to 148 in SEQ ISNO: 53) (upper sequence) and a part-sequence of the MIPS tag “Sub2” fromS. cerevisiae (lower sequence). FIG. 11B shows an alignment between anamino acid part-sequence of the invention (corresponding to the strandat position 150 to 185 in SEQ IS NO: 53) (upper sequence) and apart-sequence of the MIPS tag “Sub2” from S. cerevisiae (lowersequence). Identical sequence positions are indicated between the twosequences. Similar sequence positions are labeled with “+”.

FIG. 12 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 2 to 82 in SEQ ID NO:58 (upper sequence) and a part-sequence of the MIPS tag “DCP1” from S.cerevisiae (lower sequence). Identical sequence positions are indicatedbetween the two sequences. Similar sequence positions are labeled with“+”.

FIG. 13 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 21 to 695 in SEQ IDNO: 63) (upper sequence) and a part-sequence of the MIPS tag “PRT1” fromS. cerevisiae (lower sequence). Identical sequence positions areindicated between the two sequences. Similar sequence positions arelabeled with “+”.

FIG. 14A shows an alignment between an amino acid part-sequence of theinvention (corresponding to the strand at position 1 to 111 in SEQ IDNO: 67) (upper sequence) and a part-sequence of the MIPS tag “Rrp9” fromS. cerevisiae (lower sequence). FIG. 14B shows an alignment between anamino acid part-sequence of the invention (corresponding to the strandat position 144 to 887 in SEQ ID NO: 67) (upper sequence) and apart-sequence of the MIPS tag “Rrp9” from S. cerevisiae (lowersequence). Identical sequence positions are indicated between the twosequences. Similar sequence positions are labeled with “+”.

FIG. 15 shows an alignment between an amino acid part-sequence of theinvention (corresponding to the complementary strand at position 508 to176 in SEQ ID NO: 72) (upper sequence) and a part-sequence of the MIPStag “Rpl8b” from S. cerevisiae (lower sequence). Identical sequencepositions are indicated between the two sequences. Similar sequencepositions are labeled with “+”.

FIG. 16 shows the construction scheme for inserting an antibioticresistance cassette (G418 resistance gene under the control of theAshbya TEF promoter) behind the open reading frame (ORF) shown for“Oligo 18”.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid molecules of the invention encode polypeptides orproteins which are referred to here as proteins of the transcription,RNA processing and/or translation (for example with activity in relationto transcription, RNA processing, splicing or translation) or for shortas “TT proteins”. These TT proteins have, for example, a function in theadaptation to various growth phases and environmental and metabolicconditions such as substrates, oxygen level and the like.

Owing to the availability of cloning vectors which can be used in Ashbyagossypii, as disclosed, for example, in Wright and Philipsen (1991)Gene, 109, 99-105, and of techniques for genetic manipulation of A.gossypii and the related yeast species, the nucleic acid molecules ofthe invention can be used for genetic manipulation of these organisms,in particular of A. gossypii, in order to make them better and moreefficient producers of vitamin B2 and/or precursors and/or derivativesthereof. This improved production or efficiency may result from a directeffect of the manipulation of a gene of the invention or result from anindirect effect of such a manipulation.

The present invention is based on the provision of novel molecules whichare referred to here as TT nucleic acids and TT proteins and areinvolved in the transcription, RNA processing and/or translation, inparticular in Ashbya gossypii (e.g. in the regulation of transcription,RNA processing and/or translation). The activity of the TT molecules ofthe invention in A. gossypii influences vitamin B2 production by thisorganism. The activity of the TT molecules of the invention ispreferably modulated so that the metabolic and/or energy pathways of A.gossypii in which the TT proteins of the invention are involved aremodulated in relation to the yield, production and/or efficiency ofvitamin B2 production, which modulates either directly or indirectly theyield, production and/or efficiency of vitamin B2 production in A.gossypii.

The nucleic acid sequences provided by the invention can be isolated,for example, from the genome of an Ashbya gossypii strain which isfreely available from the American Type Culture Collection under thenumber ATCC 10895.

Improvement in Vitamin B2 Production:

There is a number of possible mechanisms by which the yield, productionand/or efficiency of production of vitamin B2 by an A. gossypii straincan be influenced directly through changing the amount and/or activityof a TT protein of the invention.

Thus, a more efficient transcription, RNA processing or translation,which adapts expression of the desired gene products to the externalconditions, can achieve optimization of the formation of the desiredproducts of value.

Mutagenesis of one or more TT proteins of the invention may also lead toTT proteins with altered (increased or reduced) activities whichinfluence indirectly the production of the required product from A.gossypii. It is possible, for example, with the aid of the TT proteinsfor the progress of transcription, RNA processing and/or translation tobe assisted (e.g by activators) or blocked (e.g. by repressors) atvarious points, and thus gene expression or protein synthesis to beinfluenced. The yield of target product can thus be increased oroptimized in relation to external conditions.

Polypeptides

The invention relates to polypeptides which comprise the abovementionedamino acid sequences or characteristic part-sequences thereof and/or areencoded by the nucleic acid sequences described herein.

The invention likewise encompasses “junctional equivalents” of thespecifically disclosed novel polypeptides.

“Functional equivalents” or analogs of the specifically disclosedpolypeptides are for the purposes of the present invention polypeptideswhich differ therefrom but which still have the desired biologicalactivity (such as, for example, substrate specificity).

“Functional equivalents” mean according to the invention in particularmutants which have in at least one of the abovementioned sequencepositions an amino acid which differs from that specifically mentionedbut nevertheless have one of the abovementioned biological activities.“Functional equivalents” thus comprise the mutants obtainable by one ormore amino acid additions, substitutions, deletions and/or inversions,it being possible for said modifications to occur in any sequenceposition as long as they lead to a mutant having the profile ofproperties of the invention. Functional equivalence exists in particularalso when there is qualitative agreement between mutant and unmodifiedpolypeptide in the reactivity pattern, i.e. there are differences in therate of conversion of identical substrates, for example.

“Functional equivalents” in the above sense are also precursors of thepolypeptides described, and functional derivatives and salts of thepolypeptides. The term “salts” means both salts of carboxyl groups andacid addition salts of amino groups in the protein molecules of theinvention. Salts of carboxyl groups can be prepared in a manner knownper se and comprise inorganic salts such as, for example, sodium,calcium, ammonium, iron and zinc salts, and salts with organic basessuch as, for example, amines such as triethanolamine, arginine, lysine,piperidine and the like. Acid addition salts such as, for example, saltswith mineral acids such as hydrochloric acid or sulfuric acid and saltswith organic acids such as acetic acid and oxalic acid are also anaspect of the invention.

“Functional derivatives” of polypeptides of the invention can also beprepared at functional amino acid side groups or at their N- orC-terminal end by known techniques. Such derivatives include, forexample, aliphatic esters of carboxyl groups, amides of carboxyl groupsobtainable by reaction with ammonia or with a primary or secondaryamine; N-acyl derivatives of free amino groups prepared by reaction withacyl groups; or O-acyl derivatives of free hydroxyl groups prepared byreaction with acyl groups. “Functional equivalents” naturally alsocomprise polypeptides which are obtainable from other organisms, andnaturally occurring variants. For example homologous sequence regionscan be found by sequence comparison, and equivalent enzymes can beestablished on the basis of the specific requirements of the invention.

“Functional equivalents” likewise comprise fragments, preferably singledomains or sequence motifs, of the polypeptides of the invention, whichhave, for example, the desired biological function.

“Functional equivalents” are additionally fusion proteins which have oneof the abovementioned polypeptide sequences or functional equivalentsderived therefrom and at least one other heterologous sequencefunctionally different therefrom in functional N- or C-terminal linkage(i.e. with negligible mutual impairment of the functions of the parts ofthe fusion proteins). Nonlimiting examples of such heterologoussequences are, for example, signal peptides, enzymes, immunoglobulins,surface antigens, receptors or receptor ligands.

“Functional equivalents” include according to the invention homologs ofthe specifically disclosed proteins. These have at least 60%, preferablyat least 75%, in particular at least 85%, such as, for example, 90%, 95%or 99%, homology to one of the specifically disclosed sequences,calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad.Sci. (USA) 85(8), 1988, 2444-2448.

In the case where protein glycosylation is possible, equivalents of theinvention include proteins of the type defined above in deglycosylatedor glycosylated form, and modified forms obtainable by altering theglycosylation pattern.

Homologs of the proteins or polypeptides of the invention can begenerated by mutagenesis, for example by point mutation or truncation ofthe protein. The term “homolog” as used here relates to a variant formof the protein which acts as agonist or antagonist of the proteinactivity.

Homologs of the proteins of the invention can be identified by screeningcombinatorial libraries of mutants such as, for example, truncationmutants. It is possible, for example, to generate a variegated libraryof protein variants by combinatorial mutagenesis at the nucleic acidlevel, such as, for example, by enzymatic ligation of a mixture ofsynthetic oligonucleotides. There is a large number of methods which canbe used to produce libraries of potential homologs from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic gene can then be ligated into a suitable expression vector.The use of a degenerate set of genes makes it possible to provide allsequences which encode the desired set of potential protein sequences inone mixture. Methods for synthesizing degenerate oligonucleotides areknown to the skilled worker (for example Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidsRes. 11:477).

In addition, libraries of fragments of the protein codon can be used togenerate a variegated population of protein fragments for screening andfor subsequent selection of homologs of a protein of the invention. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double-stranded PCR fragment of a coding sequence with anuclease under conditions under which nicking takes place only aboutonce per molecule, denaturing the double-stranded DNA, renaturing theDNA to form double-stranded DNA, which may comprise sense/antisensepairs of different nicked products, removing single-stranded sectionsfrom newly formed duplices by treatment with S1 nuclease and ligatingthe resulting fragment library into an expression vector. It is possibleby this method to derive an expression library which encodes N-terminal,C-terminal and internal fragments having different sizes of the proteinof the invention.

Several techniques are known in the prior art for screening geneproducts from combinatorial libraries which have been produced by pointmutations or truncation and for screening cDNA libraries for geneproducts with a selected property. These techniques can be adapted torapid screening of gene libraries which have been generated bycombinatorial mutagenesis of homologs of the invention. The mostfrequently used techniques for screening large gene libraries undergoinghigh-throughput analysis comprise the cloning of the gene library intoreplicable expression vectors, transformation of suitable cells with theresulting vector library and expression of the combinatorial genes underconditions under which detection of the required activity facilitatesisolation of the vector which encodes the gene whose product has beendetected. Recursive ensemble mutagenesis (REM), a technique whichincreases the frequency of functional mutants in the libraries, can beused in combination with the screening tests for identifying homologs(Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993)Protein Engineering 6(3):327-331).

Recombinant preparation of the polypeptides of the invention is possible(see following sections) or they can be isolated in native form frommicroorganisms, especially those of the genus Ashbya, by use ofconventional biochemical techniques (see Cooper, T. G., BiochemischeArbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or inScopes, R., Protein Purification, Springer Verlag, New York, Heidelberg,Berlin.

Nucleic Acid Sequences:

The invention also relates to nucleic acid sequences (single- anddouble-stranded DNA and RNA sequences such as, for example, cDNA andmRNA), coding for one of the above polypeptides and their functionalequivalents which are obtainable, for example, by use of artificialnucleotide analogs.

The invention relates both to isolated nucleic acid molecules which codefor polypeptides or proteins of the invention or biologically activesections thereof, and to nucleic acid fragments which can be used, forexample, for use as hybridization probes or primers for identifying oramplifying coding nucleic acids of the invention.

The nucleic acid molecules of the invention may additionally compriseuntranslated sequences from the 3′ and/or 5′ end of the coding region ofthe gene.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acidand may moreover be essentially free of other cellular material orculture medium if it is produced by recombinant techniques, or free ofchemical precursors or other chemicals if it is chemically synthesized.

A nucleic acid molecule of the invention can be isolated by usingstandard techniques of molecular biology and the sequence informationprovided according to the invention. For example, cDNA can be isolatedfrom a suitable cDNA library by using one of the specifically disclosedcomplete sequences or a section thereof as hybridization probe andstandard hybridization techniques (as described, for example, inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). It ismoreover possible for a nucleic acid molecule comprising one of thedisclosed sequences or a section thereof to be isolated by polymerasechain reaction using the oligonucleotide primers constructed on thebasis of this sequence. The nucleic acid amplified in this way can becloned into a suitable vector and be characterized by DNA sequenceanalysis. The oligonucleotides of the invention which correspond to a TTnucleotide sequence can also be produced by standard synthetic methods,for example using an automatic DNA synthesizer.

The invention additionally comprises the nucleic acid molecules whichare complementary to the specifically described nucleotide sequences, ora section thereof.

The nucleotide sequences of the invention make it possible to generateprobes and primers which can be used for identifying and/or cloninghomologous sequences in other cell types and organisms. Such probes andprimers usually comprise a nucleotide sequence region which hybridizesunder stringent conditions onto at least about 12, preferably at leastabout 25, such as, for example, 40, 50 or 75, consecutive nucleotides ofa sense strand of a nucleic acid sequence of the invention or acorresponding antisense strand.

Further nucleic acid sequences of the invention are derived from SEQ IDNO: 1, 4, 6, 10, 12, 14, 17, 19, 21, 24, 26, 29, 31, 36, 38, 40, 42, 46,48, 51, 53, 56, 58, 60, 63, 65, 67, 70, 72, 74, 75, or SEQ ID NO: 77 anddiffer therefrom through addition, substitution, insertion or deletionof one or more nucleotides, but still code for polypeptides having thedesired profile of properties.

The invention also encompasses nucleic acid sequences which compriseso-called silent mutations or are modified, by comparison with aspecifically mentioned sequence, in accordance with the codon usage of aspecific source or host organism, as well as naturally occurringvariants such as, for example, splice variants or allelic variants,thereof. It likewise relates to sequences which are obtainable byconservative nucleotide substitutions (i.e. the relevant amino acid isreplaced by an amino acid with the same charge, size, polarity and/orsolubility).

The invention also relates to molecules derived from the specificallydisclosed nucleic acids through sequence polymorphisms. These geneticpolymorphisms may exist because of the natural variation betweenindividuals within a population. These natural variations normallyresult in a variance of from 1 to 5% in the nucleotide sequence of agene.

The invention additionally encompasses nucleic acid sequences whichhybridize with or are complementary to the abovementioned codingsequences. These polynucleotides can be found on screening of genomic orcDNA libraries and, where appropriate, be amplified therefrom by meansof PCR using suitable primers, and then, for example, be isolated withsuitable probes. Another possibility is to transform suitablemicroorganisms with polynucleotides or vectors of the invention,multiply the microorganisms and thus the polynucleotides, and thenisolate them. An additional possibility is to synthesize polynucleotidesof the invention by chemical routes.

The property of being able to “hybridize” onto polynucleotides means theability of a polynucleotide or oligonucleotide to bind under stringentconditions to an almost complementary sequence, while there are nononspecific bindings between noncomplementary partners under theseconditions. For this purpose, the sequences should be 70-100%,preferably 90-100%, complementary. The property of complementarysequences being able to bind specifically to one another is made use of,for example, in the Northern or Southern blot technique or in PCR orRT-PCR in the case of primer binding. Oligonucleotides with a length of30 base pairs or more are normally employed for this purpose. Stringentconditions mean, for example, in the Northern blot technique the use ofa washing solution at 50-70° C., preferably 60-65° C., for example0.1×SSC buffer with 0.1% SDS (20×SSC: 3M NaCl, 0.3M Na citrate, pH 7.0)for eluting nonspecifically hybridized cDNA probes or oligonucleotides.In this case, as mentioned above, only nucleic acids with a high degreeof complementarity remain bound to one another. The setting up ofstringent conditions is known to the skilled worker and is described,for example, in Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

A further aspect of the invention relates to antisense nucleic acids.This comprises a nucleotide sequence which is complementary to a codingsense nucleic acid. The antisense nucleic acid may be complementary tothe entire coding strand or only to a section thereof. In a furtherembodiment, the antisense nucleic acid molecule is antisense to anoncoding region of the coding strand of a nucleotide sequence. The term“noncoding region” relates to the sequence sections which are referredto as 5′- and 3′-untranslated regions.

An antisense oligonucleotide may be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides long. An antisense nucleic acid ofthe invention can be constructed by chemical synthesis and enzymaticligation reactions using methods known in the art. An antisense nucleicacid can be synthesized chemically, using naturally occurringnucleotides or variously modified nucleotides which are configured sothat they increase the biological stability of the molecules or increasethe physical stability of the duplex formed between the antisense andsense nucleic acids. Examples which can be used are phosphorothioatederivatives and acridine-substituted nucleotides. Examples of modifiednucleosides which can be used for generating the antisense nucleic acidare, inter alia, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueuosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queuosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,methyl uracil-5-oxyacetate, 3-(3-amino-3-carboxypropyl)uracil, (acp3)wand 2,6-diaminopurine. The antisense nucleic acid may also be producedbiologically by using an expression vector into which a nucleic acid hasbeen subcloned in the antisense direction.

The antisense nucleic acid molecules of the invention are normallyadministered to a cell or generated in situ so that they hybridize withthe cellular mRNA and/or a coding DNA or bind thereto, so thatexpression of the protein is inhibited for example by inhibition oftranscription and/or translation.

The antisense molecule can be modified so that it binds specifically toa receptor or to an antigen which is expressed on a selected cellsurface, for example through linkage of the antisense nucleic acidmolecule to a peptide or an antibody which binds to a cell surfacereceptor or antigen. The antisense nucleic acid molecule can also beadministered to cells by using the vectors described herein. The vectorconstructs preferred for achieving adequate intracellular concentrationsof the antisense molecules are those in which the antisense nucleic acidmolecule is under the control of a strong bacterial, viral or eukaryoticpromoter.

In a further embodiment, the antisense nucleic acid molecule of theinvention is an alpha-anomeric nucleic acid molecule. An alpha-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA, with the strands running parallel to one another, incontrast to normal alpha units (Gaultier et al., (1987) Nucleic AcidsRes. 15:6625-6641). The antisense nucleic acid molecule may additionallycomprise a 2′-O-methylribonucleotide (Inoue et al., (1987) Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987)FEBS Lett. 215:327-330).

The invention also relates to ribozymes. These are catalytic RNAmolecules with ribonuclease activity which are able to cleave asingle-stranded nucleic acid such as an mRNA to which they have acomplementary region. It is thus possible to use ribozymes (for examplehammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) for the catalytic cleavage of transcripts of the inventionin order thereby to inhibit the translation of the corresponding nucleicacid. A ribozyme with specificity for a coding nucleic acid of theinvention can be formed, for example, on the basis of a cDNAspecifically disclosed herein. For example a derivative of atetrahymena-L-19 IVS RNA can be constructed, with the nucleotidesequence of the active site being complementary to the nucleotidesequence to be cleaved in a coding mRNA of the invention. (Compare, forexample, U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742).Alternatively, mRNA can be used for selecting a catalytic RNA withspecific ribonuclease activity from a pool of RNA molecules (see, forexample, Bartel, D., and Szostak, J. W. (1993) Science 261:1411-1418).

Gene expression of sequences of the invention can alternatively beinhibited by targeting nucleotide sequences which are complementary tothe regulatory region of a nucleotide sequence of the invention (forexample to a promoter and/or enhancer of a coding sequence) so thatthere is formation of triple helix structures which preventtranscription of the corresponding gene in target cells (Helene, C.(1991) Anticancer Drug Res. 6(6) 569-584; Helene, C. et al., (1992) Ann.N.Y. Acad. Sci. 660:27-36; and Maher., L. J. (1992) Bioassays14(12):807-815).

Expression Constructs and Vectors:

The invention additionally relates to expression constructs comprising,under the genetic control of regulatory nucleic acid sequences, anucleic acid sequence coding for a polypeptide of the invention; and tovectors comprising at least one of these expression constructs. Suchconstructs of the invention preferably comprise a promoter 5′-upstreamfrom the particular coding sequence, and a terminator sequence3′-downstream, and, where appropriate, other usual regulatory elements,in particular each operatively linked to the coding sequence. “Operativelinkage” means the sequential arrangement of promoter, coding sequence,terminator and, where appropriate, other regulatory elements in such away that each of the regulatory elements is able to comply with itsfunction as intended for expression of the coding sequence. Examples ofsequences which can be operatively linked are targeting sequences andenhancers, polyadenylation signals and the like. Other regulatoryelements comprise selectable markers, amplification signals, origins ofreplication and the like. Suitable regulatory sequences are described,for example, in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).

In addition to the artificial regulatory sequences it is possible forthe natural regulatory sequence still to be present in front of theactual structural gene. This natural regulation can, where appropriate,be switched off by genetic modification, and expression of the genes canbe increased or decreased. The gene construct can, however, also have asimpler structure, that is to say no additional regulatory signals areinserted in front of the structural gene, and the natural promoter withits regulation is not deleted. Instead, the natural regulatory sequenceis mutated so that regulation no longer takes place, and gene expressionis enhanced or diminished. The nucleic acid sequences may be present inone or more copies in the gene construct.

Examples of promoters which can be used are: cos, tac, trp, tet,trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PRor λ-PL promoter, which are advantageously used in Gram-negativebacteria; and the Gram-positive promoters amy and SPO2, the yeastpromoters ADC1, MFα, AC, P-60, CYC1, GAPDH or the plant promotersCaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, not or the ubiquitin orphaseolin promoter. The use of inducible promoters is particularlypreferred, such as, for example, light- and, in particular,temperature-inducible promoters such as the P_(r)P_(l) promoter. It ispossible in principle for all natural promoters with their regulatorysequences to be used. In addition, it is also possible advantageously touse synthetic promoters.

Said regulatory sequences are intended to make specific expression ofthe nucleic acid sequences possible. This may mean, for example,depending on the host organism, that the gene is expressed oroverexpressed only after induction or that it is immediately expressedand/or overexpressed.

The regulatory sequences or factors may moreover preferably influencepositively, and thus increase or reduce, expression. Thus, enhancementof the regulatory elements can take place advantageously at the level oftranscription by using strong transcription signals such as promotersand/or enhancers. However, it is also possible to enhance translationby, for example, improving the stability of the mRNA.

An expression cassette is produced by fusing a suitable promoter to asuitable nucleotide sequence of the invention and to a terminator signalor polyadenylation signal. Conventional techniques of recombination andcloning are used for this purpose, as described, for example, in T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1984) and in Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acidconstruct or gene construct is advantageously inserted into ahost-specific vector, which makes optimal expression of the genes in thehost possible. Vectors are well known to the skilled worker and can befound, for example, in “Cloning Vectors” (Pouwels P. H. et al., eds,Elsevier, Amsterdam-New York-Oxford, 1985). Vectors also mean not onlyplasmids but also all other vectors known to the skilled worker, suchas, for example, phages, viruses, such as SV40, CMV, baculovirus andadenovirus, transposons, IS elements, phasmids, cosmids, and linear orcircular DNA. These vectors may undergo autonomous replication in thehost organism or chromosomal replication.

Examples of suitable expression vectors which may be mentioned are:

Conventional fusion expression vectors such as pGEX (Pharmacia BiotechInc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), with which respectively glutathione S-transferase (GST), maltoseE-binding protein and protein A are fused to the recombinant targetprotein.

Non-fusion protein expression vectors such as pTrc (Amann et al., (1988)Gene 69:301-315) and pET 11d (Studier et al. Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)60-89).

Yeast expression vector for expression in the yeast S. cerevisiae, suchas pYepSec1 (Baldari et al., (1987) Embo J. 6:229-234), pMFα (Kurjan andHerskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for constructing vectors suitable for the use inother fungi such as filamentous fungi comprise those which are describedin detail in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Genetransfer systems and vector development for filamentous fungi, in:Applied Molecular Genetics of Fungi, J. F. Peberdy et al., eds, pp.1-28, Cambridge University Press: Cambridge.

Baculovirus vectors which are available for expression of proteins incultured insect cells (for example Sf9 cells) comprise the pAc series(Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and pVL series(Lucklow and Summers (1989) Virology 170:31-39).

Plant expression vectors such as those described in detail in: Becker,D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acids Res.12:8711-8721.

Mammalian expression vectors such as pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).

Further suitable expression systems for prokaryotic and eukaryotic cellsare described in chapters 16 and 17 of Sambrook, J., Fritsch, E. F. andManiatis, T., Molecular cloning:

A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Recombinant Microorganisms:

The vectors of the invention can be used to produce recombinantmicroorganisms which are transformed, for example, with at least onevector of the invention and can be employed for producing thepolypeptides of the invention. The recombinant constructs of theinvention described above are advantageously introduced and expressed ina suitable host system. Cloning and transfection methods familiar to theskilled worker, such as, for example, coprecipitation, protoplastfusion, electroporation, retroviral transfection and the like, arepreferably used to bring about expression of said nucleic acids in theparticular expression system. Suitable systems are described, forexample, in Current Protocols in Molecular Biology, F. Ausubel et al.,eds, Wiley Interscience, New York 1997, or Sambrook et al. MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

It is also possible according to the invention to produce homologouslyrecombined microorganisms. This entails production of a vector whichcontains at least one section of a gene of the invention or a codingsequence, in which, where appropriate, at least one amino acid deletion,addition or substitution has been introduced in order to modify, forexample functionally disrupt, the sequence of the invention (knockoutvector). The introduced sequence may, for example, also be a homologfrom a related microorganism or be derived from a mammalian, yeast orinsect source. The vector used for homologous recombination mayalternatively be designed so that the endogenous gene is mutated orotherwise modified during the homologous recombination but still encodesthe functional protein (for example the regulatory region locatedupstream may be modified in such a way that this modifies expression ofthe endogenous protein). The modified section of the TT gene is in thehomologous recombination vector. The construction of suitable vectorsfor homologous recombination is, for example, described in Thomas, K. R.and Capecchi, M. R. (1987) Cell 51:503.

Suitable host organisms are in principle all organisms which enableexpression of the nucleic acids of the invention, their allelicvariants, their functional equivalents or derivatives. Host organismsmean, for example, bacteria, fungi, yeasts, plant or animal cells.Preferred organisms are bacteria, such as those of the generaEscherichia, such as, for example, Escherichia coli, Streptomyces,Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomycescerevisiae, Aspergillus, higher eukaryotic cells from animals or plants,for example Sf9 or CHO cells. Preferred organisms are selected from thegenus Ashbya, in particular from A. gossypii strains.

Successfully transformed organisms can be selected through marker geneswhich are likewise present in the vector or in the expression cassette.Examples of such marker genes are genes for antibiotic resistance andfor enzymes which catalyze a color-forming reaction which causesstaining of the transformed cell. These can then be selected byautomatic cell sorting. Microorganisms which have been successfullytransformed with a vector and harbor an appropriate antibioticresistance gene (for example G418 or hygromycin) can be selected byappropriate antibiotic-containing media or nutrient media. Markerproteins present on the surface of the cell can be used for selection bymeans of affinity chromatography.

The combination of the host organisms and the vectors appropriate forthe organisms, such as plasmids, viruses or phages, such as, forexample, plasmids with the RNA polymerase/promoter system, phages λ or μor other temperate phages or transposons and/or other advantageousregulatory sequences forms an expression system. The term “expressionsystem” means, for example, the combination of mammalian cells, such asCHO cells, and vectors, such as pcDNA3neo vector, which are suitable formammalian cells.

If desired, the gene product can also be expressed in transgenicorganisms such as transgenic animals such as, in particular, mice, sheepor transgenic plants.

Recombinant Production of the Polypeptides:

The invention further relates to methods for the recombinant productionof a polypeptide of the invention or functional, biologically activefragments thereof, wherein a polypeptide-producing microorganism iscultured, expression of the polypeptides is induced where appropriate,and they are isolated from the culture. The polypeptides can also beproduced on the industrial scale in this way if desired.

The recombinant microorganism can be cultured and fermented by knownmethods. Bacteria can be grown, for example, in TB or LB medium and at atemperature of 20 to 40° C. and a pH of from 6 to 9. Details of suitableculturing conditions are described, for example, in T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).

If the polypeptides are not secreted into the culture medium, the cellsare then disrupted and the product is obtained from the lysate by knownprotein isolation methods. The cells may alternatively be disrupted byhigh-frequency ultrasound, by high pressure, such as, for example, in aFrench pressure cell, by osmolysis, by the action of detergents, lyticenzymes or organic solvents, by homogenizers or by a combination of aplurality of the methods mentioned.

The polypeptides can be purified by known chromatographic methods suchas molecular sieve chromatography (gel filtration), such as Q-Sepharosechromatography, ion exchange chromatography and hydrophobicchromatography, and by other usual methods such as ultrafiltration,crystallization, salting out, dialysis and native gel electrophoresis.Suitable methods are described, for example, in Cooper, T. G.,Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New Yorkor in Scopes, R., Protein Purification, Springer Verlag, New York,Heidelberg, Berlin.

It is particularly advantageous for isolation of the recombinant proteinto use vector systems or oligonucleotides which extend the cDNA byparticular nucleotide sequences and thus code for modified polypeptidesor fusion proteins which serve, for example, for simpler purification.Suitable modifications of this type are, for example, so-called tagswhich act as anchors, such as, for example, the modification known ashexa-histidine anchor, or epitopes which can be recognized as antigensby antibodies (described, for example, in Harlow, E. and Lane, D., 1988,Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Theseanchors can be used to attach the proteins to a solid support, such as,for example, a polymer matrix, which can, for example, be packed into achromatography column, or can be used on a microtiter plate or anothersupport.

These anchors can at the same time also be used for recognition of theproteins. It is also possible to use for recognition of the proteinsconventional markers such as fluorescent dyes, enzyme markers which forma detectable reaction product after reaction with a substrate, orradioactive labels, alone or in combination with the anchors forderivatizing the proteins.

The invention additionally relates to a method for the microbiologicalproduction of vitamin B2 and/or precursors and/or derivatives thereof.

If the conversion is carried out with a recombinant microorganism, themicroorganisms are preferably initially cultured in the presence ofoxygen and in a complex medium, such as, for example, at a culturingtemperature of about 20° C. or more, and at a pH of about 6 to 9 untilan adequate cell density is reached. In order to be able to control thereaction better, it is preferred to use an inducible promoter. Theculturing is continued in the presence of oxygen for 12 hours to 3 daysafter induction of vitamin B2 production.

The following nonlimiting examples describe specific embodiments of theinvention.

General Experimental Details

a) General Cloning Methods

The cloning steps carried out for the purpose of the present invention,such as, for example, restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of E. coli cells, culturing of bacteria, replication ofphages and sequence analysis of recombinant DNA, were carried out asdescribed by Sambrook et al. (1989) loc. cit.

b) Polymerase Chain Reaction (PCR)

PCR was carried out in accordance with a standard protocol with thefollowing standard mixture:

8 μl of dNTP mix (200 μM), 10 μl of Taq polymerase buffer (10×) withoutMgCl₂, 8 μl of MgCl₂ (25 mM), 1 μl of each primer (0.1 μM), 1 μl of DNAto be amplified, 2.5 U of Taq polymerase (MBI Fermentas, Vilnius,Lithuania), demineralized water ad 100 μl.

c) Culturing of E. coli

The recombinant E. coli DH5α strain was cultured in LB-amp medium(tryptone 10.0 g, NaCl 5.0 g, yeast extract 5.0 g, ampicillin 100 g/ml,H₂O ad 1000 ml) at 37° C. For this purpose, in each case one colony wastransferred, using an inoculating loop, from an agar plate into 5 ml ofLB-amp. After culturing for about 18 hours shaking at a frequency of 220rpm, 400 ml of medium in a 2 l flask were inoculated with 4 ml ofculture. Induction of P450 expression in E. coli took place after theOD578 reached a value between 0.8 and 1.0 by heat-shock induction at 42°C. for three to four hours.

-   -   d) Purification of the Required Product from the Culture

The required product can be isolated from the microorganism or from theculture supernatant by various methods known in the art. If the requiredproduct is not secreted by the cells, the cells can be harvested fromthe culture by slow centrifugation, and the cells can be lysed bystandard techniques such as mechanical force or ultrasound treatment.

The cell detritus is removed by centrifugation, and the supernatantfraction which contains the soluble proteins is obtained for furtherpurification of the required compound. If the product is secreted by thecells, the cells are removed from the culture by slow centrifugation,and the supernatant fraction is retained for further purification.

The supernatant fraction from the two purification methods is subjectedto a chromatography with a suitable resin, with the required moleculeeither being retained on the chromatography resin, or passing throughthe latter, with greater selectivity than the impurities. Thesechromatography steps can be repeated if necessary, using the same ordifferent chromatography resins. The skilled worker is proficient in theselection of suitable chromatography resins and their most effective usefor a particular molecule to be purified. The purified product can beconcentrated by filtration or ultrafiltration and be stored at atemperature at which the stability of the product is maximal.

Many purification methods are known in the art. These purificationtechniques are described, for example, in Bailey, J. E. & Ollis, D. F.Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined byprior art techniques. These comprise high performance liquidchromatography (HPLC), spectroscopic methods, staining methods, thinlayer chromatography, NIRS, enzyme assay or microbiological assays.These analytical methods are summarized in: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH:Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, pp. 575-581and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, Vol.17.

e) General Description of the MPSS Method, Clone Identification andHomology Search

The MPSS technology (Massive Parallel Signature Sequencing as describedby Brenner et al, Nat. Biotechnol. (2000) 18, 630-634; to which expressreference is hereby made) was applied to the filamentous, vitaminB2-producing fungus Ashbya gossypii. It is possible with the aid of thistechnology to obtain with high accuracy quantitative information aboutthe level of expression of a large number of genes in a eukaryoticorganism. This entails the mRNA of the organism being isolated at aparticular time X, being transcribed with the aid of the enzyme reversetranscriptase into cDNA and then being cloned into special vectors whichhave a specific tag sequence. The number of vectors with a different tagsequence is chosen to be high enough (about 1000 times higher) forstatistically each DNA molecule to be cloned into a vector which isunique through its tag sequence.

The vector inserts are then cut out together with the tag. The DNAmolecules obtained in this way are then incubated with microbeads whichpossess the molecular counterparts of the tags mentioned. Afterincubation it can be assumed that each microbead is loaded via thespecific tags or counterparts with only one type of DNA molecules. Thebeads are transferred into a special flow cell and fixed there so thatit is possible to carry out a mass sequencing of all the beads with theaid of an adapted sequencing method based on fluorescent dyes and withthe aid of a digital color camera. Although numerically high analysis ispossible with this method, it is limited by a reading width of about 16to 20 base pairs. The sequence length is, however, sufficient to make anunambiguous correlation between sequence and gene possible for mostorganisms (20 bp have a sequence frequency of ˜1×10¹²; compared withthis, the human genome has a size of “only” ˜3×10⁹ bp).

The data obtained in this way are analyzed by counting the number ofidentical sequences and comparing their frequencies with one another.Frequently occurring sequences reflect a high level of expression, andsequences which occur singly a low level of expression. If the mRNA wasisolated at two different time points (X and Y), it is possible toconstruct a chronological expression pattern of individual genes.

EXAMPLE 1

Isolation of mRNA from Ashbya gossypii

Ashbya gossypii was cultured in a manner known per se (nutrient medium:27.5 g/l yeast extract; 0.5 g/l magnesium sulfate; 50 ml/l soybean oil;pH 7). Ashbya gossypii mycelium samples are taken at various timesduring the fermentation (24 h, 48 h and 72 h), and the corresponding RNAor mRNA is isolated therefrom according to the protocol of Sambrook etal. (1989).

EXAMPLE 2

Application of the MPSS

Isolated mRNA from A. gossypii is then subjected to an MPSS analysis asexplained above.

The sets of data found are subjected to a statistical analysis andcategorized according to the significance of the differences inexpression. This entailed examination both in relation to an increaseand a reduction in the level of expression. A division is made byclassifying the change in expression into a) monotonic change, b) changeafter 24 h, and c) change after 48 h.

The 20 bp sequences representing a change in expression and found byMPSS analysis are then used as probes and hybridized with a gene libraryfrom Ashbya gossypii, with an average insert size of about 1 kb. Thehybridization temperature in this case was in the range from about 30 to57° C.

EXAMPLE 3

Construction of a Genomic Gene Library from Ashbya gossypii

To construct a genomic DNA library, initially chromosomal DNA isisolated by the method of Wright and Philippsen (Gene (1991) 109:99-105) and Mohr (1995, PhD Thesis, Biozentrum Universitat Basel,Switzerland).

The DNA is partially digested with Sau3A. For this purpose, 6 μg ofgenomic DNA are subjected to a Sau3A digestion with various amounts ofenzyme (0.1 to 1 U). The fragments are fractionated in a sucrose densitygradient. The 1 kb region is isolated and subjected to a QiaExextraction. The largest fragments are ligated to the BamHI-cut vectorpRS416 (Sikorski and Hieter, Genetics (1988) 122; 19-27) (90 ng ofBamHI-cut, dephosphorylated vector; 198 ng of insert DNA; 5 ml of water;2 μl of 10× ligation buffer; 1 U ligase). This ligation mixture is usedto transform the E. coli laboratory strain XL-1 blue, and the resultingclones are employed for identifying the insert.

EXAMPLE 4

Preparation of an Ordered Gene Library (CHIP Technology)

About 25,000 colonies of the Ashbya gossypii gene library (thiscorresponds to approximately a 3-fold coverage of the genome) weretransferred in an ordered manner to a nylon membrane and then treated bythe method of colony hybridization as described in Sambrook et al.(1989). Oligonucleotides were synthesized from the 20 bp sequences foundby MPSS analysis and were radiolabeled with ³²P. In each case 10 labeledoligonucleotides with a similar melting point are combined andhybridized together with the nylon membranes. After hybridization andwashing steps, positive clones are identified by autoradiography andanalyzed directly by PCR sequencing.

In this way, a clone which harbors an insert with the internal name“Oligo 28” and has significant homology with the MIPS tag “Yta7” or TBP7from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 1.

In this way, a further clone which harbors an insert with the internalname “Oligo 45” and has significant homology with the MIPS tag “p39” or“Tif34” from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 6.

In this way, a further clone which harbors an insert with the internalname “Oligo 85” and has significant homology with the MIPS tag “Rpl35a”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 12.

In this way, a further clone which harbors an insert with the internalname “Oligo 133” and has significant homology with the MIPS tag “Nop13”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 17.

In this way, a further clone which harbors an insert with the internalname “Oligo 172” and has significant homology with the MIPS tag “Sua5”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 21.

In this way, a further clone which harbors an insert with the internalname “Oligo 63” and has significant homology with the MIPS tag “Rps25a”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 26.

In this way, a further clone which harbors an insert with the internalname “Oligo 132” and has significant homology with the MIPS tag “Nic96”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 31.

In this way, a further clone which harbors an insert with the internalname “Oligo 174” and has significant homology with the MIPS tag “Ahcl”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 38.

In this way, a further clone which harbors an insert with the internalname “Oligo 51” and has significant homology with the MIPS tag “Rok1”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 42.

In this way, a further clone which harbors an insert with the internalname “Oligo 30” and has significant homology with the MIPS tag “Rpa34”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 48.

In this way, a further clone which harbors an insert with the internalname “Oligo 124” and has significant homology with the MIPS tag “Sub2”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 53.

In this way, a further clone which harbors an insert with the internalname “Oligo 139” and has significant homology with the MIPS tag “DCP1”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 58.

In this way, a further clone which harbors an insert with the internalname “Oligo 144” and has significant homology with the MIPS tag “PRT1”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 63.

In this way, a further clone which harbors an insert with the internalname “Oligo 168” and has significant homology with the MIPS tag “Rrp9”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 67.

In this way, a further clone which harbors an insert with the internalname “Oligo 160” and has significant homology with the MIPS tag “Rpl8b”from S. cerevisiae was identified. The insert has a nucleic acidsequence as shown in SEQ ID NO: 72.

In this way, a further clone which harbors an insert with the internalname “Oligo 18” was identified. The insert has a nucleic acid sequenceas shown in SEQ ID NO: 75 (complementary strand with SEQ ID NO: 74). Apotential ORF is located between positions 958 and 1272 shown in SEQ IDNO: 75.

EXAMPLE 5

Analysis of the Sequence Data by Means of a BLASTX Search

An analysis of the resulting nucleic acid sequences, i.e. theirfunctional assignment to a functional amino acid sequence took place bymeans of a BLASTX search in sequence databases. Almost all of the aminoacid sequence homologies found related to Saccharomyces cerevisiae(baker's yeast). Since this organism had already been completelysequenced, more detailed information about these genes could be referredto under:

http://www.mips.gsf.de/proj/yeast/search/code search.htm.

The following homologies with amino acid fragments from S. cerevisiaewere found in this way. The corresponding alignments are shown in FIGS.1 to 15.

a) Amino acid sequences (corresponding to nucleotides 3 to 374 and 373to 1479) derived from SEQ ID NO:1 have significant sequence homologieswith a 26 S proteasome subunit or the TAT-binding homolog 7 (TBP7) fromS. cerevisiae. A corresponding alignment is shown in FIG. 1. SEQ ID NO:2 and SEQ ID NO: 3 in each case show an amino acid part-sequence of theinvention.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a 26 S proteasome subunit or a TAT-binding homolog 7 (TBP7).

b) An amino acid sequence derived from SEQ ID NO: 6 (cf. SEQ ID NO: 7;corresponding to nucleotides 5 to 463 in SEQ ID NO: 6) has significantsequence homology with a translation initiation factor (EIF3) subunit(P39) from S. cerevisiae. A corresponding alignment is shown in FIG. 2.SEQ ID NO: 8 and SEQ ID NO: 9 in each case show a further amino acidpart-sequence of the invention.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a translation initiation factor subunit.

c) The amino acid sequence derived from the coding strand to SEQ ID NO:12 has significant sequence homology with a ribosomal protein from S.cerevisiae. An amino acid part-sequence derived therefrom (correspondingto nucleotides 469 to 825 from SEQ ID NO: 12) with a part-sequence ofthe S. cerevisiae protein is depicted in FIG. 3. SEQ ID NO: 13 shows anN-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a ribosomal protein.

d) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 17 has significant sequence homology with anucleolar protein from S. cerevisiae. An amino acid part-sequencederived therefrom (corresponding to nucleotides 114 to 1 from SEQ ID NO:17) with a part-sequence of the S. cerevisiae protein is depicted inFIG. 4. SEQ ID NO: 18 shows an N-terminally extended amino acidpart-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a nucleolar protein.

e) The amino acid sequence derived from the coding strand to SEQ ID NO:21 has significant sequence homology with a translation initiationprotein from S. cerevisiae. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 2 to 349 from SEQ ID NO: 21)with a part-sequence of the S. cerevisiae protein is depicted in FIG.5A. A further amino acid part-sequence derived therefrom (correspondingto nucleotides 336 to 947 from SEQ ID NO: 21) with a part-sequence ofthe S. cerevisiae protein is depicted in FIG. 5B. SEQ ID NO: 22 and SEQID NO: 23 in each case show an N-terminally extended amino acidpart-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a translation initiation protein.

f) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 26 has significant sequence homology with aprecursor of ribosomal protein S 31 from S. cerevisiae. An amino acidpart-sequence derived therefrom (corresponding to nucleotides 609 to 562from SEQ ID NO: 26) with a part-sequence of the S. cerevisiae protein isdepicted in FIG. 6A. Another amino acid part-sequence derived therefrom(corresponding to nucleotides 556 to 401 from SEQ ID NO: 26) with apart-sequence of the S. cerevisiae protein is depicted in FIG. 6B. SEQID NO: 27 and SEQ ID NO: 28 in each case show an N-terminally extendedamino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a precursor of ribosomal protein S 31.

g) An amino acid sequence derived from SEQ ID NO: 31 (cf. SEQ ID NO: 32,corresponding to nucleotides 108 to 764 in SEQ ID NO: 31) hassignificant sequence homology with a cell nuclear pore protein from S.cerevisiae. FIG. 7 shows a corresponding alignment. The sequences SEQ IDNO: 33 to SEQ ID NO: 35 show further amino acid part-sequences of theinvention.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a cell nuclear pore protein.

h) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 38 has significant sequence homology with aconstituent of the ADH-histone acetyltransferase complex from S.cerevisiae. An amino acid part-sequence derived therefrom (correspondingto nucleotides 174 to 1 from SEQ ID NO: 38) with a part-sequence of theS. cerevisiae protein is depicted in FIG. 8. SEQ ID NO: 39 shows anN-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a constituent of the ADH-histone acetyltransferase complex.

i) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 42 has significant sequence homology with an S.cerevisiae RNA helicase which is involved in RNA processing. An aminoacid part-sequence derived therefrom (corresponding to nucleotides 1086to 1012 from SEQ ID NO: 42) with a part-sequence of the S. cerevisiaeenzyme is depicted in FIG. 9A. A second amino acid part-sequence derivedtherefrom (corresponding to nucleotides 1022 to 915 from SEQ ID NO: 42)with a part-sequence of the S. cerevisiae enzyme is depicted in FIG. 9B.A further amino acid part-sequence derived therefrom (corresponding tonucleotides 925 to 689 from SEQ ID NO: 42) with a part-sequence of theS. cerevisiae enzyme is depicted in FIG. 9C. SEQ ID NO: 43, SEQ ID NO:44 and SEQ ID NO: 45 in each case show an N-terminally extended aminoacid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of an RNA helicase which is involved in RNA processing.

k) The amino acid sequence derived from the coding strand to SEQ ID NO:48 has significant sequence homology with the nonessential constituentof RNA poll from S. cerevisiae. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 1 to 102 from SEQ ID NO: 48)with a part-sequence of the S. cerevisiae protein is depicted in FIG.10A. A further amino acid part-sequence derived therefrom (correspondingto nucleotides 122 to 400 from SEQ ID NO: 48) with a part-sequence ofthe S. cerevisiae protein is depicted in FIG. 10B. SEQ ID NO: 49 and SEQID NO: 50 in each case show an amino acid part-sequence of theinvention.

The A. gossypii nucleic acid sequence found could thus be assigned afunction of the nonessential constituent of RNA poll.

l) The amino acid sequence derived from the coding strand to SEQ ID NO:53 has significant sequence homology with an RNA helicase from S.cerevisiae. An amino acid part-sequence derived therefrom (correspondingto nucleotides 2 to 148 from SEQ ID NO: 53) with a part-sequence of theS. cerevisiae enzyme is depicted in FIG. 11A. A further amino acidpart-sequence derived therefrom (corresponding to nucleotides 150 to 185from SEQ ID NO: 53) with a part-sequence of the S. cerevisiae enzyme isdepicted in FIG. 11B. SEQ ID NO: 54 and SEQ ID NO: 55 in each case showan N-terminal extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of an RNA helicase.

m) The amino acid sequence derived from the coding strand to SEQ ID NO:58 has significant sequence homology with an mRNA decapping enzyme fromS. cerevisiae. An amino acid part-sequence derived therefrom(corresponding to nucleotides 2 to 82 from SEQ ID NO: 58) with apart-sequence of the S. cerevisiae enzyme is depicted in FIG. 12. SEQ IDNO: 59 shows an N-terminally extended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of an mRNA decapping enzyme.

n) The amino acid sequence derived from the coding strand to SEQ ID NO:63 has significant sequence homology with an S. cerevisiae subunit totranslation initiation factor eIF3. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 21 to 695 from SEQ ID NO: 63)with a part-sequence of the S. cerevisiae protein is depicted in FIG.13. SEQ ID NO: 64 shows an N-terminally extended amino acidpart-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a subunit of translation initiation factor eIF3.

o) The amino acid sequence derived from the coding strand to SEQ ID NO:67 has significant sequence homology with an S. cerevisiae U3 smallnucleolar ribonucleoprotein-associated protein which is involved inpreribosomal RNA processing. An amino acid part-sequence derivedtherefrom (corresponding to nucleotides 1 to 111 from SEQ ID NO: 67)with a part-sequence of the S. cerevisiae protein is depicted in FIG.14A. A further amino acid part-sequence derived therefrom (correspondingto nucleotides 144 to 887 from SEQ ID NO: 67) with a part-sequence ofthe S. cerevisiae protein is depicted in FIG. 14B. SEQ ID NO: 68 and SEQID NO: 69 in each test show an N-terminally extended amino acidpart-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of a U3 small nucleolar ribonucleoprotein-associated proteinwhich is involved in preribosomal RNA processing.

p) The amino acid sequence derived from the corresponding complementarystrand to SEQ ID NO: 72 has significant sequence homology with aribosomal protein (L7a.e.B/large 60 S subunit) from S. cerevisiae. Anamino acid part-sequence derived therefrom (corresponding to nucleotides508 to 176 from SEQ ID NO: 72) with a part-sequence of the S. cerevisiaeprotein is depicted in FIG. 15. SEQ ID NO: 73 shows an N-terminallyextended amino acid part-sequence.

The A. gossypii nucleic acid sequence found could thus be assigned thefunction of the ribosomal protein (L7a.e.B/large 60 S subunit).

EXAMPLE 6

Isolation of Full-Length DNA

a) Construction of an A. gossypii Gene Library

High molecular weight cellular complete DNA from A. gossypii wasprepared from a 2-day old 100 ml culture grown in a liquid MA2 medium(10 g of glucose, 10 g of peptone, 1 g of yeast extract, 0.3 g ofmyo-inositol ad 1000 ml). The mycelium was filtered off, washed twicewith distilled H₂O, suspended in 10 ml of 1 M sorbitol, 20 mM EDTA,containing 20 mg of zymolyase 20T, and incubated at 27° C., shakinggently, for 30 to 60 min. The protoplast suspension was adjusted to 50mM Tris-HCl, pH 7.5, 150 mM NaCl, 100 mM EDTA and 0.5% strength sodiumdodecyl sulfate (SDS) and incubated at 65° C. for 20 min. After twoextractions with phenol/chloroform (1:1 vol/vol), the DNA wasprecipitated with isopropanol, suspended in TE buffer, treated withRNase, reprecipitated with isopropanol and resuspended in TE.

An A. gossypii cosmid gene library was produced by binding genomic DNAwhich had been selected according to size and partially digested withSau3A to the dephosphorylated arms of the cosmid vector Super-Cos1(Stratagene). The Super-Cos1 vector was opened between the two cos sitesby digestion with XbaI and dephosphorylation with calf intestinalalkaline phosphatase (Boehringer), followed by opening of the cloningsite with BamHI. The ligations were carried out in 20 μl, containing 2.5μg of partially digested chromosomal DNA, 1 μg of Super-Cos1 vectorarms, 40 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol, 0.5 mMATP and 2 Weiss units of T4-DNA ligase (Boehringer) at 15° C. overnight.The ligation products were packaged in vitro using the extracts and theprotocol of Stratagene (Gigapack II Packaging Extract). The packagedmaterial was used to infect E. coli NM554 (recA 13, araD139,Δ(ara,leu)7696, Δ(lac)17A, galU, galK, hsrR, rps(str^(r)), mcrA, mcrB)and distributed on LB plates containing ampicillin (50 μg/ml).Transformants containing an A. gossypii insert with an average length of30-45 kb were obtained.

b) Storage and Screening of the Cosmid Gene Library

In total, 4×10⁴ fresh single colonies were inoculated singly into wellsof 96-well microtiter plates (Falcon, No. 3072) in 100 μl of LB medium,supplemented with the freezing medium (36 mM K₂HPO₄/13.2 mM KH₂PO₄, 1.7mM sodium citrate, 0.4 mM MgSO₄, 6.8 mM (NH₄)₂SO₄, 4.4% (w/v) glycerol)and ampicillin (50 μg/ml), allowed to grow at 37° C. overnight withshaking, and frozen at −70° C. The plates were rapidly thawed and thenduplicated in fresh medium using a 96-well replicator which had beensterilized in an ethanol bath with subsequent evaporation of the ethanolon a hot plate. Before the freezing and after the thawing (before anyother measures) the plates were briefly shaken in a microtiter shaker(Infors) in order to ensure a homogeneous suspension of cells. A roboticsystem (Bio-Robotics) with which it is possible to transfer smallamounts of liquid from 96 wells of a microtiter plate to nylon membrane(GeneScreen Plus, New England Nuclear) was used to place single cloneson nylon membranes. After the culture had been transferred from the96-well microtiter plates (1920 clones), the membranes were placed onthe surface of LB agar with ampicillin (50 μg/ml) in 22×22 cm culturedishes (Nunc) and incubated at 37° C. overnight. Before cell confluencewas reached, the membranes were processed as described by Herrmann, B.G., Barlow, D. P. and Lehrach, H. (1987) in Cell 48, pp. 813-825,including as additional treatment after the first denaturation step a5-minute exposure of the filters to vapors on a pad impregnated withdenaturation solution on a boiling water bath.

The random hexamer primer method (Feinberg, A. P. and Vogelstein, B.(1983), Anal. Biochem. 132, pp. 6-13) was used to label double-strandedprobes by uptake of [alpha-³²P]dCTP with high specific activity. Themembranes were prehybridized and hybridized at 42° C. in 50% (vol/vol)formamide, 600 mM sodium phosphate, pH 7.2, 1 mM EDTA, 10% dextransulfate, 1% SDS, and 10× Denhardt's solution, containing salmon spermDNA (50 μg/ml) with ³²P-labeled probes (0.5-1×10⁶ cpm/ml) for 6 to 12 h.Typically, washing steps were carried out at 55 to 65° C. in 13 to 30 mMNaCl, 1.5 to 3 mM sodium citrate, pH 6.3, 0.1% SDS for about 1 h and thefilters were autoradiographed at −70° C. with Kodak intensifying screensfor 12 to 24 h. To date, individual membranes have been reusedsuccessfully more than 20 times. Between the autoradiographies, thefilters were stripped by incubation at 95° C. in 2 mM Tris-HCl, pH 8.0,0.2 mM EDTA, 0.1% SDS for 2×20 min.

c) Recovery of Positive Colonies from the Stored Gene Library

Frozen bacterial cultures in microtiter wells were scraped out usingsterile disposable lancets, and the material was streaked onto LB agarPetri dishes containing ampicillin (50 μg/ml). Single colonies were thenused to inoculate liquid cultures to produce DNA by the alkaline lysismethod (Bimboim, H. C. and Doly, J. (1979), Nucleic Acids Res. 7, pp.1513-1523).

d) Full-Length DNA

It was possible as described above to identify clones which harbor aninsert with the appropriate complete sequence. These clones have theinternal names given below:

“Oligo 28v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 4.

“Oligo 45v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 10.

“Oligo 85v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 14. The protein encoded therebypreferably comprises at least one of the amino acid sequences shown inSEQ ID NO: 15 and 16.

“Oligo 133v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 19.

“Oligo 172v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 24.

“Oligo 63v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 29.

“Oligo 132v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 36.

“Oligo 174v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 40.

“Oligo 51v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 46.

“Oligo 30v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 51.

“Oligo 124v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 56.

“Oligo 139v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 60. The protein encoded therebypreferably comprises at least one of the amino acid sequences as shownin SEQ ID NO: 61 and 62.

“Oligo 144v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 65.

“Oligo 168v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 70.

“Oligo 18v”. The insert comprising the complete sequence has a nucleicacid sequence as shown in SEQ ID NO: 77.

EXAMPLE 7

Detection of a Modulating Effect of Oligo 18 on Vitamin B2 Production

In order to test whether integration of DNA in the vicinity of thepotential reading frame of oligo 18 has adverse effects on riboflavinsynthesis, a DNA fragment was integrated by means of homologousrecombination into the genome of the Ashbya gossypii strain used (AshbyaTEF promoter+G418 resistance gene—cf. FIG. 1). Transformation took placeby electoporation in a manner known per se. Positive transformants wereidentified by PCR using the primer pair shown in FIG. 1. Onetransformant in which specific integration into this locus wasdetectable was investigated for vitamin B2 production both in shakenflask experiments and in laboratory fermentations. It emerged thatintegration of this DNA fragment brought about an increase (of about 3%)in riboflavin production. The information in the TEF-G418 constructcannot have been the reason. It is therefore concluded that there is aposition effect.

Shaken flask experiments for riboflavin determination:

10 ml of preculture medium (9.5 ml [9.5 g] of medium+0.5 ml soybean oil)in a 100-mi 2-baffle Erlenmeyer flask are inoculated with 0.5 ml of aglycerol culture or with about one inoculating loop of mycelium from a5-day old, well grown SP agar plate, and shaken at 180 rpm (cabinetshaker, excursion 2.5 cm) and 28° C. for 40 hours.

1.1 ml of this culture are used to inoculate 25.7 ml of main culturemedium (21.2 ml [21.2 g] of medium, 1 ml of urea [10 g/45 ml]+3.5 ml[3.2 g] of soybean oil, final volume=26.8 ml, of which 4.4 mlcompensates for evaporation during shaking without humidification) or21.8 ml of main culture medium (17.3 ml [17.3 g] of medium, 1 ml of urea[10 g/45 ml]+3.5 ml [3.2 g] of soybean oil, final volume=22.9 ml, ofwhich 0.5 ml compensates for evaporation during shaking in anenvironment without artificial humidification) in a 250 ml Erlenmeyerflask and shaken at 220 rpm (industrial shaker, excursion 5 cm) or 300rpm (cabinet shaker, excursion 2.5 cm) and 28° C. for 5 days.

0.5 ml of the main culture is vigorously shaken with 4.5 ml [5 g] of a40% strength nicotinamide solution (dilution factor 10) or 0.25 ml with4.75 ml [5.27 g] of a 40% strength nicotinamide solution (dilutionfactor 20) in a test tube and incubated in a water bath at 70° C. forabout 2×20 minutes (cells lyzed, shaking in between). After cooling, 40μl are put in a macro dispersible cuvette, mixed with 3 ml of deionizedwater and measured as quickly as possible in a photometer, becausevitamin B₂ decomposes very rapidly. This entails measurement of theextinctions at 402, 446 and 550 nm and calculation as follows:V=(W1−W2×C+W3×(C−1)):(B1−B2×C)with

-   B1=17.36 [constant]-   B2=31.15 [constant]-   K=cuvette volume in ml [standard=3.04 ml]-   P=sample volume in ml [standard=0.04 ml]-   F=dilution factor [standard=10, i.e. 0.5 ml: 5 ml]-   C=correction factor [(550-405)/(550-450)=1.45]-   W1=extinction at 402 nm-   W2=extinction at 446 nm-   W3=extinction at 550 nm-   ->V=(W1-1.45W2+0.45W3): -27.8075 $\begin{matrix}    {{{Vitamin}\quad B_{2}\quad{concentration}} = {V \times {K:{P \times F}}}} \\    {= {{V \times 3.04}:{0.04 \times 10}}} \\    {= {V \times 760}}    \end{matrix}$    With these values it is also necessary to take account of the    evaporation of the medium during the shaking:-   G1=weight of the flask immediately after inoculation-   G2=weight of the flask before sampling-   KV1=volume of the medium with compensation for evaporation [22.4    ml+4.4 ml=26.8 ml]-   KV2=volume of the medium [22.4 ml]-   B₂=the previously calculated, uncorrected vitamin B₂ concentration    $\begin{matrix}    {{{Vitamin}\quad B_{2}\quad{concentration}\quad({corrected})} = {\left( {\left( {{KV1} - \left( {{G1} - {G2}} \right)} \right):{KV2}} \right) \times B_{2}}} \\    {= {\left( {\left( {26.8 - \left( {{G1} - {G2}} \right)} \right):22.4} \right) \times B_{2}}}    \end{matrix}$

The A. gossypii nucleic acid sequence found could on the basis of theabove observations be assigned the function of a protein for modulatingthe vitamin B2 productivity. TABLE 1 Sequence survey SEQ ID NO: OligoDescription of the sequence Sequence homology  1 028 DNA part-sequence26 S proteasome  2 028 Amino acid part-sequence derived from subunit orTAT-binding complementary strand to SEQ ID NO: 1 homolog 7 (TBP7) from 3 028 Amino acid part-sequence derived from S. cerevisiae complementarystrand to SEQ ID NO: 1  4 028 DNA full-length sequence  5 028 Amino acidsequence corresponding to the coding region of SEQ ID NO: 4 fromposition 245 to 4222  6 045 DNA part-sequence Translation initiation  7045 Amino acid part-sequence derived from the factor subunit fromcomplementary strand to SEQ ID NO: 6 S. cerevisiae  8 045 Amino acidpart-sequence derived from the complementary strand to SEQ ID NO: 6  9045 Amino acid part-sequence derived from the complementary strand toSEQ ID NO: 6 10 045 DNA full-length sequence 11 045 Amino acid sequencecorresponding to the coding region of SEQ ID NO: 10 from position 640 to1674 12 085 DNA part-sequence Ribosomal protein from 13 085 Amino acidpart-sequence derived from the S. cerevisiae coding strand to SEQ ID NO:12 14 085 DNA full-length sequence 15 085 Amino acid sequencecorresponding to the coding region of SEQ ID NO: 14 from position 92 to307 16 085 Amino acid sequence corresponding to the coding region of SEQID NO: 14 from position 403 to 858 17 133 DNA part-sequence Nucleolarprotein from 18 133 Amino acid part-sequence derived from the S.cerevisiae complementary strand to SEQ ID NO: 17 19 133 DNA full-lengthsequence 20 133 Amino acid sequence corresponding to the coding regionof SEQ ID NO: 19 from position 1371 to 2495 21 172 DNA part-sequenceTranslation initiation 22 172 Amino acid part-sequence derived from theprotein from coding strand to SEQ ID NO: 21 S. cerevisiae 23 172 Aminoacid part-sequence derived from the coding strand to SEQ ID NO: 21 24172 DNA full-length sequence 25 172 Amino acid sequence corresponding tothe coding region of SEQ ID NO: 24 from position 277 to 1476 26 063 DNApart-sequence Ribosomal protein S 31 27 063 Amino acid part-sequencederived from the from S. cerevisiae complementary strand to SEQ ID NO:26 28 063 Amino acid part-sequence derived from the complementary strandto SEQ ID NO: 26 29 063 DNA full-length sequence 30 063 Amino acidsequence corresponding to the coding region of SEQ ID NO: 29 fromposition 533 to 856 31 132 DNA part-sequence Cell nuclear pore 32 132Amino acid part-sequence derived from the protein from complementarystrand to SEQ ID NO: 31 S. cerevisiae 33 132 Amino acid part-sequencederived from the complementary strand to SEQ ID NO: 31 34 132 Amino acidpart-sequence derived from the complementary strand to SEQ ID NO: 31 35132 Amino acid part-sequence derived from the complementary strand toSEQ ID NO: 31 36 132 DNA full-length sequence 37 132 Amino acid sequencecorresponding to the coding region of SEQ ID NO: 36 from position 629 to3181 38 174 DNA part-sequence ADH-histone 39 174 Amino acidpart-sequence derived from the acetyltransferase complementary strandSEQ ID NO: 38 complex from 40 174 DNA full-length sequence S. cerevisiae41 174 Amino acid sequence corresponding to the coding region of SEQ IDNO: 40 from position 964 to 2589 42 051 DNA part-sequence S. cerevisiaeRNA 43 051 Amino acid part-sequence derived from the helicase which iscomplementary strand to SEQ ID NO: 42 involved in RNA 44 051 Amino acidpart-sequence derived from the processing complementary strand to SEQ IDNO: 42 45 051 Amino acid part-sequence derived from the complementarystrand to SEQ ID NO: 42 46 051 DNA full-length sequence 47 051 Aminoacid sequence corresponding to the coding region of SEQ ID NO: 46 fromposition 502 to 2208 48 030 DNA part-sequence Nonessential 49 030 Aminoacid part-sequence derived from the constituent of RNA poll codingstrand to SEQ ID NO: 48 from S. cerevisiae 50 030 Amino acidpart-sequence derived from the coding strand to SEQ ID NO: 48 51 030 DNAfull-length sequence 52 030 Amino acid sequence corresponding to thecoding region of SEQ ID NO: 51 from position 198 to 1073 53 124 DNApart-sequence RNA helicase from 54 124 Amino acid part-sequence derivedfrom the S. cerevisiae coding strand to SEQ ID NO: 53 55 124 Amino acidpart-sequence derived from the coding strand to SEQ ID NO: 53 56 124 DNAfull-length sequence 57 124 Amino acid sequence corresponding to thecoding region of SEQ ID NO: 56 from position 465 to 1775 58 139 DNApart-sequence mRNA decapping 59 139 Amino acid part-sequence derivedfrom the enzyme from coding strand to SEQ ID NO: 58 S. cerevisiae 60 139DNA full-length sequence 61 139 Amino acid sequence corresponding to thecoding region of SEQ ID NO: 60 from position 402 to 638 62 139 Aminoacid sequence corresponding to the coding region of SEQ ID NO: 60 fromposition 663 to 974 63 144 DNA part-sequence Subunit of the 64 144 Aminoacid part-sequence derived from the translation initiation coding strandto SEQ ID NO: 63 factor elF3 from 65 144 DNA full-length sequence S.cerevisiae 66 144 Amino acid sequence corresponding to the coding regionof SEQ ID NO: 65 from position 468 to 2675 67 168 DNA part-sequence S.cerevisiae U3 small 68 168 Amino acid part-sequence derived from thenucleolar ribonucleo- coding strand to SEQ ID NO: 67 protein-associated69 168 Amino acid part-sequence derived from the protein which isinvolved coding strand to SEQ ID NO: 67 in preribosomal RNA 70 168 DNAfull-length sequence processing 71 168 Amino acid sequence correspondingto the coding region of SEQ ID NO: 70 from position 660 to 2432 72 160DNA part-sequence Ribosomal protein 73 160 Amino acid part-sequencederived from the (L7a.e.B; large 60 S complementary strand to SEQ ID NO:72 subunit) from S. cerevisiae 74 018 DNA part-sequence Modulator ofvitamin B2 75 018 DNA full-length sequence production 76 018 Amino acidsequence corresponding to the coding region of SEQ ID NO: 75 fromposition 958 to 1272 77 018 DNA full-length sequence 78 018 Amino acidsequence corresponding to the coding region of SEQ ID NO: 77 fromposition 1531 to 1845

1. An isolated polynucleotide that can be isolated from Ashbya gossypiiand that codes for a protein associated with transcription, RNAprocessing or translation of an organism.
 2. The polynucleotide of claim1, which has a structural or functional property comparable with aprotein selected from the group consisting of a 26S proteasome subunit,a TAT-binding homolog 7 translation initiation factor subunit, ribosomalprotein, nucleolar protein, translation initiation protein ribosomalprotein S31, cell nuclear pore protein, ADH-histone acetyltransferasecomplex RNA helicase, RNA poll, mRNA decapping enzyme subunit oftranslation initiation factor eIF3, U3 small nucleolarribonucleoprotein-associated protein ribosomal protein L7a.e.B of thelarge 60 S subunit, and a modulator of vitamin B2 production protein. 3.The polynucleotide of claim 1, comprising the sequence of SEQ ID NO: 1,6, 12, 17, 21, 26, 31, 38, 42, 48, 53, 58, 63, 67, 72 or 74; apolynucleotide complementary to said sequence; or a sequence derivedfrom said nucleic acid or said complementary polynucleotide throughdegeneracy of the genetic code.
 4. The polynucleotide in of claim 1,which comprises a nucleic acid that contains the sequence of SEQ ID NO:4, 10, 14, 19, 24, 29, 36, 40, 46, 51, 56, 60, 65, 70, 75 or 77, or afragment thereof.
 5. An isolated oligonucleotide that can hybridize tothe polynucleotide of claim
 1. 6. An isolated polynucleotide that canhybridize to the oligonucleotide of claim 5, and codes for a geneproduct derived from a microorganism of the genus Ashbya or a functionalequivalent thereof.
 7. An isolated polypeptide encoded by thepolynucleotide of claim 1 or a fragment thereof.
 8. An expressioncassette comprising the polynucleotide of claim 1 operatively linked toat least one regulatory nucleic acid sequence.
 9. A recombinant vectorcomprising at least one expression cassette of claim
 8. 10. Aprokaryotic or eukaryotic host cell transformed with the vector of claim9.
 11. The host cell of claim 10, wherein functional expression of saidpolypeptide is modulated.
 12. The host cell of claim 10 which is amicroorganism of the genus Ashbya.
 13. A method for microbiologicalproduction of vitamin B2 or a precursor or derivative thereof comprisingexpressing the polynucleotide of claim 1 in a microorganism.
 14. Amethod for recombinant production of the polypeptide of claim 7comprising expressing said polypeptide in a microorganism.
 15. A methodfor detecting an effector target for modulating microbiologicalproduction of vitamin B2or a precursor or derivative thereof, comprisingtreating a microorganism capable of the microbiological production ofvitamin B2 or a precursor or derivative thereof with an effector thatinteracts with a target wherein said target comprises the polypeptide ofclaim 7 or a nucleic acid that encodes said polypeptide and detectingsaid effector target.
 16. A method for modulating microbiologicalproduction of vitamin B2 or a precursor or derivative thereof comprisingtreating a microorganism capable of the microbiological production ofvitamin B2 or a precursor or derivative thereof with an effector thatinteracts with a target wherein said target comprises the polypeptide ofclaim 7 or a nucleic acid that encodes said polypeptide.
 17. An isolatedeffector selected from the group consisting of: antibodies orantigen-binding fragments thereof that bind to the polypeptide of claim7; polypeptide ligands that are different from said antibodies andantigen-binding fragments and that interact with said polypeptide; lowmolecular weight effectors that modulate a biological activity of saidpolypeptide; antisense nucleic acid sequences, catalytic RNA moleculesand ribozymes which interact with a nucleic acid sequence that encodessaid polypeptide; and combinations and mixtures thereof.
 18. A methodfor microbiological production of vitamin B2 or a precursor orderivative thereof, comprising: culturing the host cell of claim 10 in aculture mixture under conditions favoring the microbiological productionof vitamin B2 or the precursor or derivative thereof; and isolating aproduct from the culture mixture.
 19. The method of claim 18, whereinthe host cell is treated with an effector before or during culturing.20. The method of claim 18, wherein the host cell is a microorganism ofthe genus Ashbya.
 21. A method for modulating production of vitamin B2or a precursor or derivative thereof in a microorganism of the genusAshbya comprising treating said microorganism with the polynucleotide ofclaim
 1. 22. A method for modulating production of vitamin B2 or aprecursor or derivative thereof in a microorganism of the genus Ashbyacomprising treating said microorganism with the polypeptide of claim 7and hereby modulating production as desired.
 23. A method for modulatingtranscription, RNA processing or translation of a microorganism of thegenus Ashbya comprising culturing said microorganism for microbiologicalproduction of vitamin B2 or a precursor or derivative thereof with thepolynucleotide of claim 1 or with a polypeptide encoded by saidpolynucleotide.
 24. The host, cell of claim 12, which has an improvedadaptability to an environmental or a metabolic condition as comparedwith an untransformed cell that provides said cell with an improvedproduction of vitamin B2 or a precursor or derivative thereof.
 25. Thepolynucleotide of claim 1, wherein the organism is A. gossypii or S.cerevisiae.
 26. The polynucleotide of claim 1, wherein the protein isassociated with transcription, translation, or RNA processing of theorganism.
 27. The polynucleotide of claim 2, wherein the protein isderived from a microorganism of A. gossypii or S. cerevisiae.
 28. Theoligonucleotide of claim 5, wherein hybridization is under stringentconditions.
 29. The polynucleotide of claim 6, wherein hybridization isunder stringent conditions.
 30. An isolated polypeptide or fragmentthereof encoded by the polynucleotide of claim
 6. 31. An isolatedpolypeptide or fragment thereof which has an amino acid sequence thatcomprises at least ten consecutive amino acid residues of SEQ ID NO: 2,3, 5, 7, 8, 9, 11, 13, 15, 16, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33,34, 35, 37, 39, 41, 43, 44, 45, 47, 49, 50, 52, 54, 55, 57, 59, 61, 62,64, 66, 68, 69, 71, 73, 76 or 78; or a functional equivalent thereof.32. The polypeptide of claim 31, which has an activity comparable with aprotein selected from the group consisting of a 26S proteasome subunit,a TAT-binding homolog 7, translation initiation factor subunit,ribosomal protein, nucleolar protein, translation initiation protein,ribosomal protein S31, cell nuclear pore protein, ADH-histoneacetyltransferase complex, RNA helicase, RNA poll, mRNA decappingenzyme, subunit of translation initiation factor eIF3, U3 smallnucleolar ribonucleoprotein-associated protein, ribosomal proteinL7a.e.B of the large 60 S subunit, and a modulator of vitamin B2production protein.
 33. The polypeptide of claim 32, wherein the proteinis derived from a microorganism of A. gossypii or S. cerevisiae.
 34. Thehost cell of claim 10, wherein biological activity of said protein isreduced or increased.
 35. The method of claim 11, wherein modulatingcomprises an increase or decrease in the functional expression of saidprotein.
 36. The method of claim 13, wherein expressing said polypeptideresults in an improved production of vitamin B2 or a precursor orderivative thereof by said microorganism.
 37. The method of claim 36,wherein the improved production comprises an increased yield, productionor efficiency of production by said microorganism.
 38. The method ofclaim 15, wherein detecting validates said effector target.
 39. Themethod of claim 15, where the effector binds to said target.
 40. Themethod of claim 15, further comprising isolating said target.
 41. Themethod of claim 19, wherein the effector is selected from the groupconsisting of: antibodies or antigen-binding fragments thereof that bindto a polypeptide associated with transcription, translation or RNAprocessing of A. gossypii; polypeptide ligands that are different fromsaid antibodies or antigen-binding fragments and that interact with saidpolypeptide; low molecular weight effectors that modulate a biologicalactivity of said polypeptide; antisense nucleic acid sequences,catalytic RNA molecules and ribozymes which interact with a nucleic acidsequence that encodes said polypeptide; and combinations and mixturesthereof.
 42. The method of claim 21, wherein modulating comprises anincrease in rate or amount of the vitamin B2 or the precursor orderivative thereof produced by said microorganism.
 43. The method ofclaim 22, wherein modulating comprises an increase in rate or amount ofthe vitamin B2 or the precursor or derivative thereof produced by saidmicroorganism.
 44. A recombinant cell with a modified transcription,translation or RNA processing that provides for an increased productionof vitamin B2, or a precursor or derivative thereof, as compared with anon-recombinant cell.
 45. The recombinant cell of claim 37, which is A.gossypii or S. cerevisiae.